Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo.
J Gene Med
; 14(5): 328-38, 2012 May.
Article
en En
| MEDLINE
| ID: mdl-22438271
ABSTRACT
BACKGROUND:
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors.METHODS:
Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR.RESULTS:
Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF.CONCLUSIONS:
We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Regulación de la Expresión Génica
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Silenciador del Gen
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MicroARNs
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Factor A de Crecimiento Endotelial Vascular
Tipo de estudio:
Prognostic_studies
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Risk_factors_studies
Límite:
Animals
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Humans
Idioma:
En
Revista:
J Gene Med
Asunto de la revista:
BIOLOGIA MOLECULAR
/
GENETICA MEDICA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Dinamarca